Method of preparing Limulus amoebocyte lysate

ABSTRACT

Disclosed is a method of preparing limulus amoebocyte lysate substantially free from factor G which comprises bringing limulus amoebocyte lysate into contact with an insoluble carrier on which a (1→3)-β-D-glucoside structural portion represented by the following formula [I] produced by depolymerizing and/or fractionating a carbohydrate chain is immobilized: ##STR1## wherein n represents an integer of 2 to 370.

SUMMARY OF THE INVENTION

The present invention relates to a method of preparing limulusamoebocyte lysate.

BACKGROUND OF THE INVENTION

It has been widely known that limulus amoebocyte lysate (hereinafterreferred to as LAL) reacts with an endotoxin which is a bacterialpyrogen (hereinafter referred to as endotoxin) to cause a coagulation.

On the basis of this reaction, various methods of assaying endotoxinhave been developed.

Recently, the above-described coagulation reaction mechanism has beenelucidated, i.e., coagulogen is converted to coagulin to cause acoagulation (gelation) according to a stepwise reaction as shown in FIG.1 [S. Iwanaga et al., The hemolymph coagulation system in invertebrateanimals, J. Protein Chem., 5, 255-268 (1986)]. It can be understood thatthe reaction mechanism includes two coagulation systems: a systeminitiated by endotoxin (factor C system) and a system initiated by(1→3)-β-D-glucan (e.g., curdlan, partially carboxyfnethylated(1→3)-β-D-glucan) (factor G system).

The present inventors already found that the (1→3)-β-D-glucan structuralportion having a particular molecular weight inhibits the activation ofthe system of LAL initiated by (1→3)-β-D-glucan (factor G system), andfiled the patent application as a limulus amoebocyte lysate factor Gactivation inhibitor (Japanese Patent Application No. 63-216341 and WO90/02951).

However, when this inhibitor is added to LAL in order to obtain LALspecific to endotoxin, a complex comprising the inhibitor and factor Gremaining in LAL is possibly dissociated with a sample added and theliberated factor G is activated by a factor G-activating substance.

DETAILED DESCRIPTION OF THE INVENTION

An object of the present invention is to provide LAL free from factor Gwhich is first activated with (1→3)-β-D-glucan in the LAL coagulationmechanism.

Thus, the present invention provides a method of preparing limulusamoebocyte lysate substantially free from factor G which comprisescontacting limulus amoebocyte lysate with an insoluble carrier on which(1→3)-β-D-glucoside structural portion having formula [I] is fixed.##STR2## wherein n represents an integer of 2 to 370.

The (1→3) -β-D-glucoside structural portion used in the presentinvention is a polyglycoside having, in one molecule, at least onepoly-(1→3)-β-D-glucoside structural portion [hereinafter referred to asa poly(1→3)glucoside moiety] comprising 2 to 370, preferably 3 to 310,and more preferably 4 to 180 consecutive (1→3)-β-D-glucoside structuralunits (molecular weight: 162) represented by the following formula.##STR3##

Thus, the polyglycoside used in the present invention is required tohave at least one poly(1→3)glucoside moiety in one molecule. Forexample, the polyglycoside used in the present invention may consistssubstantially of one poly(1→3)glucoside moiety, for example,poly-(1→3)-β-D-glucoside represented by the following formula ##STR4##wherein n is an integer of 2 to 370, preferably 3 to 310, and morepreferably 4 to 180. Alternatively, it may be of a structure in whichone poly(1→3)glucoside moiety has a carbohydrate chain, which may bebound to the above-described poly(1→3)glucoside moiety as a branchedchain, composed of one or more (1→4)-β-D-glucoside structural unitsrepresented by the following formula; ##STR5## one or more (1→6)-β-D-glucoside structural unit represented by the following formula;##STR6## or one or more modified β-D-glucoside structural unitrepresented by the following formulae; ##STR7## wherein at least one ofR₁, R₂ and R₃ represent(s) a chemically introducible group(s), i.e., amethyl group, a hydroxyalkyl group such as a hydroxymethyl group, acarboxyalkyl group such as a carboxymethyl group, an acetyl group, asulfate group, a phosphate group, or an allyl group, a metal salt of anyof the above-mentioned groups, an ammonium salt and an organic aminesalt of any of the above-mentioned groups, and the remaining groupsrepresent a hydrogen atom.

Further, the polyglycoside used in the present invention may have astructure in which two or more of the above-described poly(1→3)glucosidemoieties are linked to each other via the other carbohydrate moieties asshown by the following formula

    A.sub.1 --B.sub.1 --A.sub.2 --B.sub.2 . . .

wherein each of A₁, A₂, . . . represents a poly(1→3)-β-D-glucosidemoiety having 2 to 370, preferably 3 to 310 and, more preferably 4 to180 (1→3)-β-D-glucoside structural units represented by the aboveformula (I) continuously bound to one another, the number of the unitsof formula (I) constituting each moiety of A₁, A₂, . . . may bedifferent from each other, and B₁, B₂, . . . each represent the same ordifferent carbohydrate chain moieties as set forth above. The othercarbohydrate chain structural portions represented by B₁, B₂, . . . maybe, for example, a structural moiety composed of one structural unitrepresented by the above-described formula (II), (III), (IV), (V) or(VI) or two or more such structural units.

Furthermore, the polyglycoside used in the present invention may havesuch a structure that the above-described poly(1→3)glucoside moiety islinked to a long-chain poly(1→3)-β-D-glucoside structural portioncomposed of 371 or more consecutive (1→3)-β-D-glucoside structural unitsrepresented by the above-described formula (I), via the othercarbohydrate chain structures as shown by the above-described B₁, B₂, .. .

Accordingly, the polyglycoside used in the present invention contains atleast one of the above-described poly(1→3)glucoside moieties permolecule, and its molecular weight is not particularly limited.

Further, it is preferred that the polyglycoside used in the presentinvention substantially comprises at least one above-describedpoly(1→3)glucoside moiety per molecule, but it should not be limitedthereto. For example, it may also contain other polyglycosides includinga high molecular weight poly(1→3)-β-D-glucoside moiety havingconsecutive 371 or more (1→3)-β-D-glucoside structural units representedby the above-described formula (I). This is because the polyglycosideaccording to the present invention is more rapidly and more stronglybound to factor G which is an initiation factor of a factor G activationsystem of LAL, than a high molecular weight poly(1→3)-β-D-glucosidewhich is a factor G activating substance used to thereby inhibit theactivation of factor G to an activated factor G. Therefore, the presenceof such high molecular weight poly(1→3)-β-D-glucoside does notsubstantially affect the inhibition action of the polyglycoside of thepresent invention.

The molecular weight of the polyglycoside used herein is determined byperforming gel permeation chromatography using a standard substance ofknown molecular weight under the following conditions to prepare acalibration curve, subjecting a test sample to gel permeationchromatography under the same conditions, and comparing the results withthe calibration curve.

Column: TSKgel G-PWXL series (Tosoh Corporation), 7.8×300 mm severalcolumns of several types,

Mobile phase: 0.3M NaOH

Flow rate: 0.5 ml/min

Sample solution concentration: 0.1 to 5 mg/ml

Sample solution volume injected: 0.1 ml

Column temperature: room temperature

Detection method: measurement by a differential refractometer (LKB Co.)or quantitative analysis of carbohydrate by the phenol-sulfuric acidmethod

Standard substance: TSK standard polyethylene oxide (Tosoh Corporation)and polyethylene glycol (Nacalai Tesque), 10 types having an averagemolecular weight ranges 1,000 to 860,000

The polyglycosides of the present invention as defined above may bederived from natural sources or may be synthesized. They also may bepartially chemically modified products of poly(1→3)-β-D-glucoside havingthree or more (1→3)-β-D-glucoside structural units represented by theabove-described formula (I). Usually, those derived from natural sourcesare readily available. Examples of such polyglycoside are describedbelow.

(1) substantially straight-chain polyglucosides consisting substantiallyof the (1→3)-β-D-glucoside structural units represented by the aboveformula (I) such as (1→3)-β-D-glucans derived from bacteria of the genusAlcaligenes, paramylon derived from flagellates Euglena, β-glucans fromfibrous tissues of higher plants or callose extracted from sieve tubesof higher plants, D-glucose polymers having (1→3)-β-bonds and containedin partially hydrolyzed products of laminatans derived from brown algaeof the genus Laminaria and Eisenia or the above-described(1→3)-β-D-glucan, laminaridextrins having a polymerization degree of 10to 20, laminarioligosaccharides having a polymerization degree of 10 orless, etc.

(2) Polyglycosides comprising the (1→3)-β-D-glucoside structural unitsrepresented by the above formula (I) and the (1→6)-β-D-glucosidestructural units represented by the above formula (III) exemplified by:

a) polyglycosides having a main carbohydrate chain of (1→3)-β-bondsincorporating one to several glucoses connected by the (1→6)-β-bond, forexample, laminatans derived from brown algae of the genus Eisenia;

b) polyglycosides as described in above a), in which a carbohydratechain of the (1→3)-β-bonds is linked to the glucose or the glucosepolymers as a branched chain through the (1→6)-β-bond, which may furtherpartially include other carbohydrate portions, for example, laminaransderived from brown algae of the genus Laminaria, chrysolaminaransderived from diatoms such as Ochromonas, Phaeodactylum, Skeletonema,Biddulphia, Coscinodiscus, and Chaetoceros and pachyman derived fromPoria;

c) polyglycosides having many more branches and being dendriform such asβ-glucan contained in the cell walls of Ascomycetes, Basidiomycetes andPhycomycetes, for example, glucans derived from the cell wall ofPhytophthora, etc.;

d) polyglycosides having a straight chain (1→3)-β-glucan to whichglucose is linked through the (1→6)-β-bond, for example, sclerotanderived from Sclerotinia having a glucosyl branch on every threeglucosyl residues of the main chain, schizophytlan derived fromSchizophyllum, grifolan LE derived from Grifola frondosa, scleroglucansderived from Sclerotium, Corticium and Stromatinia, etc. or those havinga straight chain (1→3)-β-glucan to which glucoses are linked through the(1→6)-β-bond at a rate of two glucosyl residues per five glucosylresidues of the main chain, for example, lentinan derived from Lentinus,etc., and

e) polyglycosides having a straight chain (1→6)-β-glucan having pluralglucose branches from the C-3 position of glucose residues of the mainchain through the (1→3)-β-bond, for example, β-glucan derived from thecell wall of Saccharomyces (bakers' yeast), etc.

(3) Polyglycosides having both the (1→3)-β-D-glucoside structural unitsrepresented by the above formula (I) and the (1→4)-β-D-glucosidestructural units represented by the above formula (II), for example,lichenans derived from Cetraria, Usnea, Evernia, etc., β-glucanscontained in barley endosperm, etc., which are composed of acarbohydrate chain comprising oligoglucosides linked to each otherthrough the (1→3)-β-bond and having (1→3)-β-oligoglucosides atintervals.

Some of the above-described polyglycosides are commercially availableand can be used as is. If necessary, the polyglycosides are usedfollowing a partial decomposition and/or a fractionation treatment toprepare a fraction rich in a polyglycoside containing the(1→3)-β-D-glucoside structural unit represented by the above formula (I)in the above specified amount.

Such partial decomposition and fractionation treatment can be effectedaccording to a known method. For example, partial decomposition ofcarbohydrate chains can be effected by hydrolysis with an acid, analkali or β-glucanase, acetolysis, sonication or the like. A molecularweight fractionation can be effected by precipitation with an organicsolvent such as alcohol, acetone and ether, or salts, or a fractionationusing a molecular sieve agent or a molecular sieve membrane.

Further, a part of the carbohydrate chains of the polyglycoside asexemplified in the above (1) to (3) may be chemically modified with analkyl group such as a methyl group, a hydroxyalkyl group such as ahydroxymethyl group, a carboxyalkyl group such as a carboxymethyl group,an acidic group such as an acetyl group, a sulfate group or a phosphategroup, or other functional groups. They can be prepared by introducingthese functional groups according to a known method [for example, (1)Seikagakukenkyuho (Methods of Studying Biochemistry) I, edited by Ando,Terayama, Nishizawa and Yamakawa, 284-303 (1967), Asakura Shoten, (2)Whistler, R. L. ed.: Methods in Carbohydrate Chemistry III, 193-267,271-331 (1964), Academic Press, etc.]. Particularly, (1→3)-β-D-glucanhaving a molecular weight of about 60,000 or more and having factor Gactivating action becomes usable if it is subjected to a partialchemical modification so as to adjust the number of the(1→3)-β-D-glucoside structural units represented by the above formula(I) in the poly(1→3)-β-D-glucoside moiety to 370 or less.

Thus, suitable examples of the polyglycosides of the present inventionare as follows:

Laminarioligosaccharide having a molecular weight of 342 to 1,638;

Laminaridextrin having a molecular weight of 1,800 to 3,258;

(1→3)-β-D-glucan having an average molecular weight of 2,000 to 60,000;

Laminaran having an average molecular weight of 3,000 to 23,000;

Sclerotan having an average molecular weight of 3,000 to 20,000;

Schizophyllan having an average molecular weight of 500,000 or less;

Lentinan having an average molecular weight of 1,100,000 or less;

Bakers' yeast-derived water-soluble glucan having an average molecularweight of 12,000 or less;

Lichenan having an average molecular weight of 33,000 or less;

Barley β-glucan having an average molecular weight of 200,000 or less;

Partially carboxymethylated (1→3)-β-D-glucan (substitution degree: 0,003to 1.0) having an average molecular weight of 40,000 to 240,000 obtainedby, for example, a partial carboxymethylation of curdian and saltsthereof;

Partially carboxymethylated laminaran (substitution degree: 1.0 or less)having an average molecular weight of 23,000 or less and salts thereof;

Partially methylated (1→3)-β-D-glucan (substitution degree: 0.003 to1.0) having an average molecular weight of 80,000 or less;

Partially sulfated laminaran (substitution degree: 1.0 or less) havingan average molecular weight of 23,000 or less and salts thereof.

Any insoluble carrier can be used for immobilization of theabove-described (1→3)-β-D-glucoside structural portion having thepoly(1→3)glucoside moiety as long as it has hydrophilic groups such as ahydroxy group and a carbamoyl group. Examples of these insolublecarriers are as follows: cellulose such as cellulose powder (availablefrom Advantec Toyo), Cellulofine (available from Seikagaku Corporation),Avicel (available from Funakoshi Pharmaceutical), Cellex (available fromBio-Rad); agarose such as Sepharose (available from Pharmacia), Biogel A(available from Bio-Rad ), Chromagel A (available from DojindoLaboratories), Sagavac (available from Seravac Laboratories), Gelarose(available from Litex), P-L Agarose (available from P-L Biochemicals);cross-linked dextran such as Sephadex G and Sephacryl (available fromPharmacia), P-L Dex (available from P-L Biochemicals); polyacrylamidesuch as Biogel P (available from BioRad), Chromagel P (available fromDojindo Laboratories); porous glass such as Bioglass (available fromBio-Rad); hydrophilic polyvinyl synthetic polymer such as Toyopearl(available from Tosoh).

For immobilizing the (1→3)-β-D-glucoside structural portion on theseinsoluble carriers, these carriers are required to be activated. Theremay be mentioned various activation methods including, for a carrierhaving hydroxyl groups, a cyanogen bromide method (R. Axen, J. Porath,and S. Ernback, Nature, 214, 1302 (1967)) and a method using oxiranes(J. Porath and N. Fornstedt, J. Chromatogr., 51, 479 (1970) and L.Sundberg and J. Porath, J. Chromatogr., 90, 87 (1974)), for a carrierhaving carbamoyl groups, a method using an alkyl diamine to convert thecarrier to an aminoalkylamine derivative, and a method using hydrazineto convert the carrier to a hydrazine derivative (both described in J.K. Inman and H. M. Dintzis, Biochemistry, 8, 4074 (1969)). In view ofstability and decreased nonspecific absorption, excellent is a methodwhich comprises epoxy-activating a carrier with epichlorohydrin orbisoxiranes, reacting the resulting epoxy-activated insoluble carrierwith hydrazine hydrate or dihydrazide compound to obtain a hydrazinederivative or a dihydrazide derivative, which serves as an activatedproduct (Isamu Matsumoto et al. JP-A-59-15401).

LAL used in the method according to the present invention is notparticularly limited as long as it is extracted from limulus bloodcorpuscles and the factor C system thereof is activated in a reactionwith endotoxin. Usable as such LAL include commercially availableproducts such as a freeze-dried product of LAL, a freeze-dried productof LAL with a synthetic substrate and the like.

Various commercially available lysate are as follows: Pregel, Pregel-S,Pregel-M, Pyrodick, Toxicolor (all available from SeikagakuCorporation), Limulus II-Test Wako, Limulus II-single Test Wako, LimulusHS II-Test Wako, Limulus HS II-single Test Wako, Limulus S II-singleTest Wako, Limulus amoebocyte lysate II (freeze-dried product), Limulusamoebocyte lysate-HS II (freeze-dried product) (all available from WakoPure Chemical Industries), Pyrotell (available from Cape Cod), Pyrosate(available from Haemachem), Pyrogent®, pyrogent® Plus, Pyrogent® SingleTest, Pyrogent® Multitest, LAL Single Test Kit, QCL-1000, Kinetic QCL™(all available from Whittaker Bioproducts), Coatest® Endotoxin (KabiBitrum).

The method of preparing the (1→3)-β-D-glucoside structural portion usedin the present invention is further illustrated in more detail below.

The (1→3)-β-D-glucoside structural portion used in the present inventioncan be prepared, for example, by the method described in the followingpreparation examples. Commercially available (1→3)-β-D-glucan productsfallen within the scope of the present invention can be used as it is.

PREPARATION EXAMPLE 1 Preparation from commercially available curdlan bymeans of molecular sieve chromatographic-fractionation

One gram of curdlan (Wako Pure Chemical Industries, Lot No. PEQ 9080,Mn>136,000, Mw/Mn>2.76, sample. No. 101) was dissolved in 0.3M NaOH (5mg/ml). Each 100 microliter aliquot of the resulting solution wassubjected to gel permeation chromatography (hereinafter abbreviated asGPC) at room temperature under the following conditions. {Columns:TSKgel G6000PW_(XL) and G5000PW_(XL) (both 7.8×300 mm) are connected inseries, mobile phase: 0.3M NaOH, flow rate: 0.5 ml/min}. The eluted lowmolecular fraction (Nos. 44 to 46) was collected, which was then furthersubjected to a chromatography to obtain 0.015 mg of a sample (sampleNo. 1) having the number average molecular weight of 3,050 and thepolydispersity of 1.29. The above-described GPC fraction pattern isshown in FIG. 2. Fraction patterns obtained by a rechromatography offraction Nos. 44 to 46 in FIG. 2 are shown in FIG. 3.

The present sample No. 1 was digested with β-1,3-glucanase(Zymolyase-100T, available from Seikagaku Corporation), then, theresulting digest was analyzed by means of GPC (columns: TSKgelG4000PW_(XL), G3000PW_(XL) and G2500PW_(XL) connected in series; mobilephase: distilled water, flow rate: 0.6 ml/min), whereby it could beconfirmed that a sugar composition in the digest was glucose 40%,laminaribiose 30%, laminaritriose 20%, laminaritetraose 8%,laminaripentaose 2%, recovery 94%. Thus, it can be seen that the sugarstructure of the present sample (No. 1) is β-polyglucoside comprisingthe (1→3)-β-D-glucoside structural portion.

PREPARATION EXAMPLE 2 Fractionation of curdlan depending onwater-solubility

Fifty grams of a commercially available curdlan (sample No. 101) wassuspended in a distilled water, and fractionated according to theprocedure shown in the following flow sheet. ##STR8##

PREPARATION EXAMPLE 3 Preparation of water-insoluble carbohydratefraction of curdlan by formic acid decomposition

45 g of the sample No. 102 was subjected to formic acid decompositionaccording to a method of K. Ogawa et al. [Carbohydr. Res., 29., 397-403(1973)]. The procedure is shown in the following flow sheet. ##STR9##

PREPARATION EXAMPLE 4-1 Refractionation of water-soluble fraction offormic acid decomposition product of curdian by molecular sieves

0.15 g of the water-soluble fraction obtained in the above-describedPreparation Example 3 (sample No. 3) was dissolved in 30 ml of distilledwater, and subjected to GPC (columns: TSKgel G3000PW_(XL) ×2,G2500PW_(XL)× 1, mobile phase: distilled water, flow rate: 0.5 ml/min)to collect 0.5 ml fractions. Subsequent rechromatography gave sixsamples (Nos. 11 to 16) each having a different molecular weight.

PREPARATION EXAMPLE 4-2 Refractionation of water-insoluble fraction offormic acid decomposition product of curdlan by molecular sieves

0.2 g of the water-insoluble fraction obtained in Preparation Example 3(sample No. 4) was dissolved in 40 ml of 0.3M NaOH solution, andfractionated in the same manner as in the above-described PreparationExample 4-1 using GPC (columns: TSKgel G3000PW_(XL) ×2, G25000PW_(XL)×1, mobile phase: 0.3M NaOH solution, flow rate: 0.5 ml/min), followedby rechromatography. The resulting eluate was neutralized by adding 0.3MHCl solution to obtain two samples each having a different molecularweight (Nos. 17 and 18).

PREPARATION EXAMPLE 5 Preparation of sample from water-insolublefraction of curdlan by sonication

One gram of the sample No. 102 was suspended in about 100 ml of 5 mMNaOH solution and the suspension was subjected to sonication for 12minutes at 20 KHz and 80 W under ice-cooling using Sonicator™ (Model5202PZT, Ohtake Works, Tokyo) to lower the molecular weight of thesample.

A 5M NaOH solution was added to the resulting suspension so as to give afinal concentration of 0.3M NaOH solution. Then, fractionation bychromatography was carried out in the same manner as in theabove-described Preparation Example 4-2 to obtain eight samples eachhaving a different molecular weight (Nos. 19 to 22 and 103 to 106).

PREPARATION EXAMPLE 6-1 Preparation of inhibitor derived from seaweed(I)

A sample derived from Eisenia bicyclis was prepared as follows inaccordance with the method by T. Usui et al., (Agric. Biol. Chem. 43,603-611, (1979)). 100 g of a commercially available Eisensia bicyclisdried frond (Suita Syoten, Tokyo) was pulverized and subjected toextraction with 80% ethanol to remove a low molecular weightwater-soluble fraction. Then, from the resulting residue, a laminaranfraction was extracted using 2% aqueous CaCl₂ solution. 95% ethanol wasadded to the extract to give a final concentration of 75%, and theformed precipitate was collected by centrifugation. After washing withethanol, a crude laminaran sample was obtained. It was then dissolved indistilled water and treated with an anion exchanger (DEAE-Toyopearl) toremove contaminating acidic substances (alginic acid, etc.) andpigments. Reprecipitation was effected with ethanol to obtain sample No.25.

PREPARATION EXAMPLE 6-2 Preparation of inhibitor derived from seaweed(II)

A sample derived from Laminaria japonica was prepared as follows inaccordance with the method described in J. J. Cormell et al., J. Chem.Soc., 3494 (1950). Thus, 100 g of a commercially available Laminariajaponica dried frond (Suita Syoten, Tokyo) was pulverized, a 0.09M HClsolution was added thereto and it was allowed to stand for about threedays to effect extraction. Insoluble matters were removed by afiltration and the resulting filtrate was allowed to stand for furtherone day. A small amount of the precipitate formed was removed bycentrifugation. To the supernatant obtained was added 3-fold volume ofethanol so as to give a final concentration of 75%. The precipitate thusformed was collected by centrifugation, washed with alcohol and dried toobtain a water-soluble laminaran fraction (sample No. 27).

PREPARATION EXAMPLE 7-1 Preparation of inhibitor derived from Eumycetes(I)

A sclerotan sample derived from Eumycetes Sclerotinia libertiana wasprepared as follows in accordance with the method described in Kitaharaet al., Res. Bull. Fac. Agric. Gifu University 8, 100-105 (1957). Thus,the defatted dry powder of sclerotium of Sclerotinia libertiana (30 g)was subjected to extraction with water. The resulting residue wasfurther subjected to extraction with a 7% NaOH solution and a 10% CuSO₄solution was added to the extract to form precipitate. The resultingprecipitate was collected by filtration, washed with hydrochloricacid-acidic methanol to remove copper and then washed with 80% methanolto remove HCl. The residue was purified by three repetitions of washingwith methanol and ether and drying to obtain 6 g of sample No. 28.

PREPARATION EXAMPLE 7-2 Preparation of inhibitor derived from Eumycetes(II)

A sample derived from Eumycetes Schizophyllum commune was prepared fromcommercially available schizophyllan (Kaken Chemical, trade name:Sonifilan, medicine Lot No. J61040) in accordance with the methoddescribed in K. Tabata et al., Carbohydr. Res., 89, 121-135, (1981).Thus, an aqueous solution of schizophyllan was sonicated for 10 hours inthe same manner as in the procedure of the above Preparation Example 5,followed by molecular sieve fractionation under an alkaline condition toobtain three samples different in molecular weight (Nos. 29, 30 and 31).

PREPARATION EXAMPLE 7-3 Preparation of inhibitor derived from Eumycetes(III)

A β-glucan sample derived from yeast Saccharomyces cerevisiae (bakers'yeast) was prepared as follows. Thus, 50 ml of distilled water was addedto 90 mg of a commercially available bakers' yeast glucan (Sigma, LotNo. 56F-4027) followed by stirring at room temperature for 2 hours.After centrifugation, about 50 ml of the supernatant was concentratedunder a reduced pressure to 1 ml. Insoluble matters were removed bycentrifugation. Thus, 0.64 g of sample No. 33 was obtained from thesupernatant.

PREPARATION EXAMPLE 8 Preparation of sample derived from barley β-glucan

A commercially available barley β-glucan (Sigma, Lot No. 56F-0652) wasdissolved in a 0.3M NaOH solution to give a concentration of 5 mg/ml.Then, in accordance with the method described in Preparation Example4-2, the solution was subjected to molecular sieve fractionation underan alkaline condition to obtain the β-glucan sample (No. 36) having anarrow molecular weight distribution.

Further, the above-described commercially available β-glucan wasdissolved in a hot water to give a concentration of 5 mg/ml, followed bycentrifugation (3,500 rpm, 10 minutes). The supernatant thus obtainedwas subjected to GPC fractionation (100 μl per each run, 50 runs) usingdistilled water as a mobile phase in accordance with the methoddescribed in Preparation Example 4-1. The fractionation was repeatedunder the same conditions to obtain two samples different in molecularweight (sample Nos. 37 and 38).

PREPARATION EXAMPLE 9 Preparation of partially carboxymethylated(1→3)-β-D-glucan (degree of substitution DS =0.63)

A curdlan water-insoluble product obtained according to PreparationExample 2 was carboxymethylated in accordance with the method describedin A. E. Clarke and B. A. Stone: Phytochemistry 1, 175-188 (1962). Thus,100 g of a curdlan water-insoluble product was dissolved in 1 l of a 5MNaOH solution at 0° C. in nitrogen gas stream. To the resulting solutionwas added dropwise 236 g of monochloroacetic acid dissolved in 200 ml ofwater with stirring. After the completion of the addition, stirring wascontinued for 2 hours at 60° to 65° C. The formed gel was pulverized byvigorous stirring in a 2.5-fold volume of ethanol followed byfiltration. The resulting residue was thoroughly washed successivelywith 70% ethanol, ethanol and ether, and dried. The dried product wasdissolved in 7 l of water and neutralized with 1M acetic acid. Then, 40g of an activated carbon was added thereto and the resulting solutionwas stirred at room temperature for 1 hour followed by filtration. Thefiltrate was concentrated under a reduced pressure to 1 l, and 3-foldvolume of ethanol was added thereto to form precipitate. The precipitatewas washed with ethanol and ether and dried over concentrated sulfuricacid under reduced pressure to obtain 113.85 g of a desired product.

The partially carboxymethylated (1→3)-β-D-glucan thus obtained wasmeasured for the etherification degree (Degree of Substitution: DS) inaccordance with the uranyl nitrate method of D. F. Durso (Methods inCarbohydrate Chem. VIII, 127-129 (1980). As a result, it was found to be0.63, which means that 0.63 residue out of substitutable three hydroxylgroups in one glucose residue constituting a carbohydrate chain wassubstituted.

25 mg of the resulting partially carboxymethylated (1→3)-β-D-glucan wasdissolved in 5 ml of 0.1M ammonium acetate solution and fractionated bymeans of GPC (column: Toyopearl HW65F, 5×100 cm, mobile phase: 0.1Mammonium acetate solution, flow rate: 5.8 ml/min). The fraction thusobtained was further subjected to GPC using different columns (columns:TSKgel G6000PW_(XL) +G5000PW_(XL) +G3000PW_(XL) connected in series,mobile phase: 0.1M ammonium acetate solution, flow rate: 0.6 ml/min) toobtain sample No. 41 having a narrow molecular weight distribution(Mn=231,000).

Further, 0.3 g of the partially carboxymethylated (1→3)-β-D-glucan wasdissolved in 30 ml of distilled water and the resulting solution wassonicated (9 kHz, 180-130 W, 1 hour, a sonicator, Insonator Model 201manufactured by Kubota Works) for molecular weight reduction.

To a 4.5 ml portion of the resulting solution was added 0.5 ml of a 1Mammonium acetate solution. After mixing, fractionation by GPC andrefractionation by GPC were effected in the same manner as the procedurefor obtaining sample No. 41. Thus, two samples (Nos. 39 and 40)different in molecular weight were obtained.

PREPARATION EXAMPLE 10 Preparation of partially carboxymethylated(1→3)-β-D-glucan having a degree of substitution of 1.2

Ten grams of carboxymethylated (1→3)-β-D-glucan having the substitutiondegree (DS) of 0.63 obtained in Preparation Example 9 was added to 25 mlof a 10.5M NaOH solution at 0° C in a nitrogen gas stream to form apaste. To the resulting paste was added a monochloroacetic acid solution(10 g/12 ml ) with thoroughly stirring. The mixture was heated to 60° C.and stirred for 4 hours. After cooling, 30 ml of 2M HCl was addedthereto, which was then poured into 200 ml of hydrochloric acidicethanol (40 ml HCl/ethanol). The precipitate thus formed wassuccessively washed with 70% ethanol, ethanol and ether and dried underreduced pressure to obtain sample No. 107.

The degree of substitution was measured in the same manner as the methodof measuring the partially carboxymethylated (1→3 )-β-D-glucan havingthe DS of 0.63 obtained in Preparation Example 9. As a result, the DS ofthe sample was 1.20

PREPARATION EXAMPLE 11 Preparation of partially carboxymethylatedlaminaran

A partially carboxymethylated laminaran was prepared from laminaranderived from Laminaria digitata (Sigma, Lot No. 77F-3885) as in thepartial carboxymethylation method in Preparation Example 9, according tothe method described in A. E. Clarke and B. A. Stone: Phytochem. 1, 175(1962) to obtain sample No. 42 (DS=0.06)

PREPARATION EXAMPLE 12 Preparation of partially methylated(1→3)-β-D-glucan

According to the method described in M. Samec, Kolloid-Beihefte 51, 369(1940), 3.0 g of curdlan water-insoluble product obtained in PreparationExample 2 was suspended in 80 ml of water, to which 1.35 ml of saturatedaqueous NaOH solution was added in a nitrogen gas stream to completelydissolve the curdlan water-insoluble product. 60 g of dimethyl sulfatewas gradually added thereto at 4° C. After about one hour, the reactionmixture was added dropwise to acetone and the formed precipitate wascollected. The precipitate was thoroughly washed with acetone and driedover concentrated sulfuric acid under reduced pressure to obtain 3.13 gof the above-described product (sample No. 43, DS=0.16).

PREPARATION EXAMPLE 13 Preparation of partially sulfated laminaran

Laminaria digitata-derived laminaran was sulfated in pyridine using apyridine-sulfur trioxide complex (Wako Pure Chemical Industries, Lot No.PPL 8823) as described below.

0.5 g of thoroughly dried laminaran derived from Laminaria digitata(Sigma, Lot No. 77F-3885) was dissolved in 50 ml of dehydrated pyridine,1 g of pyridine-sulfur trioxide complex was added thereto, and then, areaction was effected at 60° C. for 1 hour. 100 ml of water was added tothe resulting reaction mixture and the mixture was cooled followed byneutralization with NaOH. The neutralized solution was dialyzed againstwater using a dialysis membrane (Spectropore 1,000 cut) which hadpreviously washed well with an aqueous alkaline solution to removeglucan. After the dialysate was concentrated, twice volume of acetonewas added thereto allow a carbohydrate component to precipitate. Theprecipitate was washed with acetone and dried over concentrated sulfuricacid under reduced pressure to obtain 0.38 g of the above-describedproduct (sample No. 44, DS=0.14).

A degree of substitution of methyl group(s) and sulfate group(s) in eachproduct obtained in Preparation Examples 12 and 13 was determined andcalculated according to the methods described in the following articles(1) and (2).

(1)- Ochiai, Tsuda, Sakamoto: Organic quantitative analysis(microanalysis), Nanzando (1956),

(2) Whistler, R. L. ed., Methods in Carbohydrate Chemistry III, pp.229-235, 227-280 (1964 ), Academic Press.

Commercially available samples

The following commercially available samples were, after determinationof their physicochemical properties, subjected directly or afteralkali-solubilization and neutralization to measurements.

Glucose: (WaKo Pure Chemical Industries, JIS guaranteed special gradereagent), sample No. 108

Laminarioligosaccharides: (Seikagaku Corporation, pure reagent) sampleNos. 5 to 10

Laminaran: derived from Eisenia araborea (Nacalai Tesque reagent),sample No. 23

Laminaran: derived from E. araborea (Tokyo Kasei Kogyo, reagent), sampleNo. 24

Laminaran: derived from Laminaria digitata (Sigma, reagent), sample No.26

Lentinan: derived from Lentinus edodes (Aginomoto, medicine Lot No.9Z01LS), sample No. 32

Lichenan: derived from Cetraria islandica (Sigma, reagent), sample No.34

Lichenan: derived from Usnea barbara (Sigma, reagent), sample No. 35

Determination results of molecular weights, factor G activationinhibitory titer, etc. of the above-described samples are shown inTable-1 below.

                                      TABLE 1                                     __________________________________________________________________________                                                     Factor G                                                                      Activation                                                                    Inhibitory                   Sample                         Carbohydrate      Titer                        No. Substance  Preparation Method                                                                            Structure.sup.1)                                                                     Mn.sup.2)                                                                           Mw/Mn                                                                              (unit/mg)                    __________________________________________________________________________     1  Curdlan GPC fraction                                                                     Preparation Example 1                                                                         (1)     3,050                                                                              1.29 1,000,000                     2  Curdlan water-soluble                                                                    Preparation Example 2                                                                         (1)     3,270                                                                              2.49 2,240,000                        product                                                                       Curdlan formic acid                                                           decomposition product                                                      3  Water-soluble                                                                            Preparation Example 3                                                                         (1)     2,080                                                                              1.90 10,700,000                       fraction                                                                   4  Water-insoluble                                                                          Preparation Example 3                                                                         (1)    10,000                                                                              3.19 324,000                          fraction                                                                   5  Laminaribiose                                                                            Commercially available product                                                                (1)      342        214                                       (Seikagaku Corporation)                                         6  Laminaritriose                                                                           Commercially available product                                                                (1)      504       4,670                                      (Seikagaku Corporation)                                         7  Laminaritetraose                                                                         Commercially available product                                                                (1)      667      20,000                                      (Seikagaku Corporation)                                         8  Laminaripentaose                                                                         Commercially available product                                                                (1)      829      39,800                                      (Seikagaku Corporation)                                         9  Laminarihexaose                                                                          Commercially available product                                                                (1)       991     55,000                                      (Seikagaku Corporation)                                        10  Laminariheptaose                                                                         Commercially available product                                                                (1)     1,153     103,000                                     (Seikagaku Corporation)                                            Curdlan formic acid                                                           decomposition product                                                     11  GPC fraction 1                                                                           Preparation Example 4-1                                                                       (1)     2,370                                                                              1.20 708,000                      12  GPC fraction 2                                                                           Preparation Example 4-1                                                                       (1)     3,400                                                                              1.20 13,400,000                   13  GPC fraction 3                                                                           Preparation Example 4-1                                                                       (1)     4,800                                                                              1.20 20,000,000                   14  GPC fraction 4                                                                           Preparation Example 4-1                                                                       (1)     5,800                                                                              1.20 31,600,000                   15  GPC fraction 5                                                                           Preparation Example 4-1                                                                       (1)     6,800                                                                              1.20 6,310,000                    16  GPC fraction 6                                                                           Preparation Example 4-1                                                                       (1)     9,800                                                                              1.22 3,980,000                    17  GPC fraction 7                                                                           Preparation Example 4-2                                                                       (1)    14,500                                                                              1.24 1,820,000                    18  GPC fraction 8                                                                           Preparation Example 4-2                                                                       (1)    27,500                                                                              1.26 126,000                          Curdlan sonicated                                                             product                                                                   19  GPC fraction 1                                                                           Preparation Example 5                                                                         (1)    20,700                                                                              1.27 646,000                      20  GPC fraction 2                                                                           Preparation Example 5                                                                         (1)    28,300                                                                              1.18 389,000                      21  GPC fraction 3                                                                           Preparation Example 5                                                                         (1)    50,200                                                                              1.26  4,900                       22  GPC fraction 4                                                                           Preparation Example 5                                                                         (1)    58,100                                                                              1.29   234                            Laminaran from                                                            23  Eisenia araborea                                                                         Commercially available                                                                        .sup.  (2)a)                                                                         16,800                                                                              1.49  6,760                                      product (Nacalai Tesque)                                       24  Eisenia araborea                                                                         Commercially available                                                                        .sup.  (2)a)                                                                         11,200                                                                              1.55 29,500                                      product (Tokyo Kasei)                                          25  Eisenia bycyclis                                                                         Preparation Example 6-1                                                                       .sup.  (2)a)                                                                         22,500                                                                              1.27 64,600                       26  Laminaria digitata                                                                       Commercially available                                                                        .sup.  (2)b)                                                                          5,850                                                                              1.16 7,080,000                                   product (Sigma)                                                27  Laminaria japonica                                                                       Preparation Example 6-2                                                                       .sup.  (2)b)                                                                         17,700                                                                              3.98 39,800                       28  Sclerotan  Preparation Example 7-1                                                                       .sup.  (2)d)                                                                         16,800                                                                              2.77 26,300                           Shizophyllan from                                                         29  GPC fraction 1                                                                           Preparation Example 7-2                                                                       .sup.  (2)d)                                                                          6,750                                                                              3.14 138,000                      30  GPC fraction 2                                                                           Preparation Example 7-2                                                                       .sup.  (2)d)                                                                         23,600                                                                              2.37 11,700                       31  GPC fraction 3                                                                           Preparation Example 7-2                                                                       .sup.  (2)d)                                                                         27,500                                                                              1.49 50,100                       32  Lentinan   Commercially available                                                                        .sup.  (2)d)                                                                         94,700                                                                              1.46 10,000                                      product (Aginomoto)                                            33  Bakers' yeast glucan                                                                     Preparation Example 7-3                                                                       .sup.  (2)e)                                                                         11,600                                                                              5.14 11,500                           water-soluble product                                                         Lichenan from                                                             34  Cetraria islandica                                                                       Commercially available product                                                                (3)    22,000                                                                              4.72  3,550                                      (Sigma)                                                        35  Usnea barbata                                                                            Commercially available product                                                                (3)    23,200                                                                              4.07   120                                       (Sigma)                                                            Barley β-glucan                                                      36  GPC fraction 1                                                                           Preparation Example 8                                                                         (3)    54,900                                                                              1.16 30,900                       37  GPC fraction 2                                                                           Preparation Example 8                                                                         (3)    129,000                                                                             1.09 11,700                       38  GPC fraction 3                                                                           Preparation Example 8                                                                         (3)    200,000                                                                             1.13 40,700                           Partially carboxy-                                                            methylated (1→3)-β-                                               D-glucan (DS = 0.03)                                                      39  GPC fraction 1                                                                           Preparation Example 9                                                                         (1)    42,400                                                                              1.14 117,000                      40  GPC fraction 2                                                                           Preparation Example 9                                                                         (1)    77,300                                                                              1.10 91,200                       41  GPC fraction 3                                                                           Preparation Example 9                                                                         (1)    231,000                                                                             1.10 80,000                       42  Partially carboxy-                                                                       Preparation Example 11                                                                        .sup.  (2)b)                                                                          8,170                                                                              1.21 3,630,000                        methylated laminaran                                                      43  Partially methylated                                                                     Preparation Example 12                                                                        (1)    78,200                                                                              1.10 93,300                           (1→3)-β-D-glucan                                              44  Partially sulfated                                                                       Preparation Example 13                                                                        .sup.  (2)b)                                                                         10,300                                                                              2.04 117,000                          laminaran                                                                 101 Curdlan    Commercially available product                                                                (1)     136,000<                                                                            2.76<                                                                                100>                                     (Wako Pure Chemical Industries)                                102 Curdlan water-                                                                           Preparation Example 2                                                                         (1)     159,000<                                                                            2.50<                                                                                100>                          insoluble product                                                             Curdlan sonicated                                                             product                                                                   103 GPC fraction 5                                                                           Preparation Example 5                                                                         (1)    76,300                                                                              1.26    100>                      104 GPC fraction 6                                                                           Preparation Example 5                                                                         (1)    92,600                                                                              1.23    100>                      105 GPC fraction 7                                                                           Preparation Example 5                                                                         (1)    171,000                                                                             1.19    100>                      106 GPC fraction 8                                                                           Preparation Example 5                                                                         (1)    216,000                                                                             1.19    100>                      107 Partially carboxy-                                                                       Preparation Example 10                                                                        (1)     329,000<                                                                            1.27<                                                                                100>                          methylated (1→3)-β-                                               D-glucan (DS = 1.20)                                                      108 Glucose    Commercially available product                                                                         180         100>                      __________________________________________________________________________     .sup.1) The number for the sugar chain structure is the classification        number defined in the specification.                                          .sup.2) Regarding glucose and laminarioligosaccharides, Mn represents an      absolute molecular weight (theoretical value). Mn for others is calculate     in terms of polyethylene oxide and polyethylene glycol according to the       separately described molecular weight determination method.              

The molecular weights in the table are represented in terms ofnumber-average molecular weights (Mn) defined by the following formuladetermined by means of the above-described gel permeation chromatography(hereinafter, sometimes abbreviated as GPC). A molecular weightdistribution is represented by the polydispersity (Mw/Mn) defined by thefollowing formula; ##EQU1##

In the above formula, Hi represents the i-th peak height (sampleconcentration) when a chromatogram is equally divided by time, and Mirepresents the i-th molecular weight.

The factor G activation inhibitory titer was measured according to thefollowing activation titer determination method of factor G activationinhibiting substance and represented as units per mg. Activation titerdetermination method of factor G activation inhibiting substance(hereinafter, sometimes abbreviated as GI)

A 200 μl portion of a reaction mixture has the following composition.

    ______________________________________                                        (1) Sample (note 1): GI sample or 50 μl pure                                   water [with or without addition of 10 pg of                                   factor G activating substance (abbreviated as                                 GA, note 2)]                                                              (2) Limulus amoebocyte lysate 30     μl                                        proclotting enzyme                                                            fraction (A.sub.280 = 2.5) (note 3)                                       (3) Limulus amoebocyte lysate factor                                                                        20     μl                                        G fraction (A.sub.280 = 0.9) (note 3)                                     (4) Tris-hydrochloric acid buffer                                                                           20     μmole                                     (pH 8.0)                                                                  (5) MgCl.sub.2                20     μmole                                 (6) Boc-Leu-Gly-Arg-pNA       0.13   μmole                                 ______________________________________                                    

After incubating the above-described reaction mixture at 37° C. for 30minutes, 0.5 ml each of 0.04% sodium nitrite (0.48M HCl solution), 0.3%ammonium sulfamate and 0.07% N-1-naphthylethylenediamine dihydrochloridewas added to the reaction mixture to effect coloration bydiazo-coupling. The amount of released pNA was determined by measuringan absorbance at 545 nm (A₅₄₅). GI activity was calculated according tothe following equation; ##EQU2##

Under these conditions, the GI amount which causes 100% inhibition of anactivation of factor G by GA is defined as 100 units.

(Note 1) Water-insoluble samples are dissolved in 0.3M NaOH andneutralized with an equal volume of 0.3M HCl.

(Note 2) GPC fractionation-purified product of the curdian sonicatedproduct prepared in the above-described Preparation Example 5 (Table-2,No. 106, molecular weight 216,000).

(Note 3) Prepared from Japanese horseshoe crab, T. tridentatus accordingto the article [T. Obayasi et al., Clin. Chim. Acta., 149, 55-65(1985)].

The insoluble fixation product of the present invention obtained byfixing the above-described (1→3)-β-D-glucoside structural portion on aninsoluble carrier is then contacted with LAL. The contact can be carriedout at 0° to 40° C., preferably 0° to 10° C., and at a pH of 6 to 8.Successively, the insoluble immobilized product is separated from LAL.The separation can be conducted by subjecting the mixture to filtrationor centrifugation to remove the insoluble fixation product, or byapplying LAL on a column packed with the insoluble immobilized productto obtain the passed LAL.

Since the amount of the insoluble immobilized product for contactingwith LAL varies depending upon a strength of factor G activationinhibitory activity of the (1→3)-β-D-glucoside structural portion fixedon a carrier, it can be determined, for example, as follows. Underice-cooling, various amount of the insoluble immobilized product(comprising no endotoxin) are contacted with a predetermined amount ofLAL, then the insoluble immobilized product is removed by acentrifugation, to which is added a predetermined amount of a factor Gactivating substance (comprising no endotoxin and comprising a factor Gactivation inhibiting substance in an amount as small as possible) whichcan substantially activates LAL under ordinary determination conditions,followed by reaction under the same conditions as those usually employedwhen LAL is used. The amount of the insoluble immobilized product whichshows 100% inhibition of an activation of LAL is determined under theabove-described conditions. Then the insoluble immobilized product inthe amount determined in the above is contacted with the above-describedpredetermined amount of LAL, successively, various amounts of the factorG activating substance are further added, whereby it is confirmed thatLAL is not activated with any amount of the factor G activatingsubstance. According to the above-described procedure, the amount of theinsoluble immobilized product which is required to completely inhibitthe activation of factor G in a certain amount of LAL can be determined.

The preparation of the insoluble immobilized product of the presentinvention as well as the preparation of LAL which specifically reactswith endotoxin utilizing the same is described by way of the followingexamples. The insoluble immobilized product was prepared according tothe method of Matsumoto et al. (Isamu Matsumoto et al., JP-A-59-15401).

PREPARATION EXAMPLE 14 Preparation of cellulose having formic aciddecomposition product (Mn, 5,800) of curdlan immobilized thereon

Two grams of cellulose powder (100-200 mesh, manufactured by Toyo Roshi)was thoroughly washed with water on a glass filter, followed by suctionfiltration. The residue thus obtained was placed into a flask, and 30 mlof water, 13 ml of a 2M NaOH solution and 3 ml of epichlorohydrin weresuccessively added thereto. The resulting suspension was agitated at 40°C. for 2 hours, and then thoroughly washed on a glass filter to obtainepoxy-activated cellulose. To the thus-obtained epoxy-activatedcellulose (20 ml) were added 1.5 volumes (30 ml) of a 80% aqueoushydrated hydrazine solution followed by agitation at 40° C. for 1.5hours. After the reaction, the resulting product was thoroughly washedwith water on a glass filter to obtain hydrazino-cellulose. To 2 g (wetweight) of the thus-obtained hydrazinocellulose were added 1.5 ml of a0.2M K₂ HPO₄ solution in which 50 mg of curdlan formic aciddecomposition product obtained in Preparation Example 4-1 (sample No.14, Mn =5,800) and 26 mg of sodium cyanoborohydride were dissolved,followed by agitation at room temperature for three days. After thereaction, the resulting product was successively washed with 1 ml ofwater and 1 ml of an aqueous 0.2M sodium acetate solution on a glassfilter. One milliliter of an aqueous 0.2M sodium acetate solution wasadded thereto to form a suspension and 0.5 ml of acetic anhydride wasfurther added to the resulting suspension. After the reaction at 0° C.for 30 minutes, 0.5 ml of acetic anhydride was further added thereto andthe resulting mixture was allowed to stand at room temperature for 30minutes to thereby acetylate unreacted hydrazine residual groups. Afterthe reaction, the resulting product was successively washed with water,a 0.1M NaOH solution, water and phosphate-buffered saline (PBS) toobtain cellulose having curdtan formic acid decomposition productimmobilized thereon.

PREPARATION EXAMPLE 15 Preparation of laminaran-immobilized cellulose

20 g (wet weight) cellulose (Cellulofine, GC-700-m, available fromSeikagaku Corporation) was converted to a hydrazine derivative in thesame manner as in Preparation Example 14. To 2 g (wet weight) of thethus-obtained hydrazinocellulofine was added 1.5 ml of an aqueous 0.2MK₂ HPO₄ solution in which 50 mg of laminaran derived from Laminariadigitata (available from Sigma, Lot No. 77F-3885) and 26 mg of sodiumcyanoborohydride were dissolved, followed by agitation at roomtemperature for three days. After the reaction, the resulting productwas washed, treated for acetylation of unreacted hydrazine residualgroups and washed again according to the method of Preparation Example14. Thus, laminaran-immobilized Cellulofine was obtained.

PREPARATION EXAMPLE 16 Preparation of laminaribiose-immobilizedsynthetic hydrophilic polyvinyl polymer

One kilogram (wet weight) of Toyopearl (synthetic hydrophilic polyvinylpolymer, HW55, Fine, available from Tosoh) was treated in the samemanner as in Preparation Example 14 to obtain epoxy-activated Toyopearl.

To 800 ml of the resulting epoxy-activated Toyopearl was added 1.2 l ofa 0.1M Na₂ CO₃ solution containing 92 g of adipic acid dihydrazidedissolved therein which had been adjusted to pH 9 with hydrochloricacid, followed by agitation overnight at 40° C. After the reaction, theresulting product was thoroughly washed on a glass filter with a 0.2MNaCl solution to obtain hydrazide Toyopearl. To the total amount of theresulting hydrazide Toyopearl was added 600 ml of a 0.2M K₂ HPO₄solution in which 32 g of laminaribiose (available from SeikagakuCorporation, Lot 8701100) and 10.4 g of sodium cyanoborohydride weredissolved, followed by agitation at room temperature for three days.After the reaction, the resulting product was successively washed withwater and 0.2M sodium acetate solution on a glass filter. Thereto wereadded 400 ml of 0.2M sodium acetate solution and 200 ml of aceticanhydride, followed by agitation at 0° C. for 30 minutes. 200 ml ofacetic anhydride was further added to the resulting suspension, whichwas agitated at room temperature for 30 minutes to thereby acetylateunreacted hydrazide residual groups. After the reaction, the resultingproduct was washed successively with water, a 0.1M NaOH solution, waterand phosphate-buffered saline. Thus, laminaribiose-immobilized Toyopearlwas obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reaction mechanism of a limulus amoebocyte lysatecoagulation system.

FIG. 2 shows a fraction pattern (elution curve) of a commerciallyavailable curdlan obtained by a molecular sieve chromatography. Thesymbol ◯ stands for factor G activation inhibition titer (left side,ordinate axis) and Δ stands for a sugar content.

FIG. 3 shows a fraction pattern of rechromatographed fraction Nos. 44 to46 of FIG. 2. The symbol ◯ stands for factor G activation inhibitiontiter (left side, ordinate axis) and Δ stands for a carbohydratecontent.

FIG. 4A shows absorbance obtained when untreated LAL or LAL treated inaccordance with the present invention was reacted with variousconcentrations of endotoxin.

The symbol ◯ stands for untreated LAL and Δ stands for LAL treated inaccordance with Example 2.

FIG. 4B shows absorbance obtained when untreated LAL or LAL treated inaccordance with the present invention was reacted with variousconcentrations of (1→3)-β-D-glucan. The symbol ◯ stands for untreatedLAL and Δ stands for LAL treated in accordance with Example 2.

EXAMPLES EXAMPLE 1 Preparation of LAL using formic acid decompositionproduct of curdlan immobilized on cellulose

Cellulose having formic acid decomposition product of curdlanimmobilized thereon (wet volume 0.4 ml) obtained in Preparation Example14 was successively washed with 1 l of 0.1M NaOH and 1 l of distilledwater to eliminate endotoxin. Distilled water was added thereto to make2.6 ml of a suspension. 1 vial of Pregel-M main agent (Limulus testproduct for gelation method, freeze-dried product, Lot AB01, sensitivity0.125 EU/ml, available from Seikagaku Corporation) was dissolved in thesuspension, which was centrifuged at 3,000 rpm for 10 minutes to obtaina supernatant (LAL-1). Reactivities of the supernatant thus obtained andPregel-M main agent dissolved in 2.6 ml of distilled water (LAL-2) toendotoxin (derived from E. coli 0111:B4) and to partiallycarboxymethylated (1→3)-β-D-glucan (Sample No. 41 obtained inPreparation Example 9, hereinafter referred to as (1→3)-β-D-glucan) wasexamined by checking the presence or absence of gelation according to astandard method for Pregel-M (0.1 ml of LAL was added to 0.1 ml of asample and the mixture was allowed to stand at 37° C. for 60 minutes.The results are shown in Table-2 in which the symbols + and - meanpresence and absence of gelation, respectively.

                  TABLE 2                                                         ______________________________________                                        Endotoxin                                                                             0       12.5   25     50    100   200 pg/ml                           ______________________________________                                        LAL-1   --      --     --    ++   ++    ++                                    LAL-2   --      --     --    ++   ++    ++                                    ______________________________________                                        (1→3)-β-D-                                                        glucan  0.1      1.0   10    100  1,000 10,000 ng/ml                          ______________________________________                                        LAL-1   --      --     --    --   --    --                                    LAL-2   --      --     ++    ++   --    --                                    ______________________________________                                    

As is apparent from the Table, LAL-1 is desirably reactive with onlyendotoxin.

EXAMPLE 2 Preparation of LAL using laminaran-immobilized cellulose(Cellulofine)

0.4 ml (wet volume) of laminaran-immobilized Cellulofine obtained inPreparation Example 15 was successively washed with 1 l of 0.1M NaOH and1 l of distilled water. Distilled water was added thereto to make thetotal volume 4 ml. To 0.2 ml of the same was added 1.8 ml of distilledwater to form a suspension, which was filtered using a polyvinylidenefluoride membrane (0.22 μm, Millex GV filter unit, available from NihonMillipore Ltd., Lot CE11) to obtain a membrane to whichlaminaran-immobilized Cellulofine was adhered. Separately, limulus(Tachypleus tridentatus) hemolymph was collected and subjected tocentrifugation (3,000 rpm, 5 minutes) to obtain hemocytes (about 20 g).100 ml of 0.02M Tris-HCl buffer (pH 8.0) was added thereto and themixture was homogenized with Waring blender, followed by centrifugation(8,000 rpm, 30 minutes, to separate supernatant and precipitate. Thisextraction procedure was repeated once again and 150 ml of thesupernatant in all was obtained to serve as LAL. A 1 ml portion of theresulting LAL was filtered using the above-described membrane to whichlaminaran-immobilized Cellulofine was adhered to obtain a filtrate. To a0.04 ml portion of the filtrate were added 1.5 μg of MgCl₂ and 4.0 μg ofsynthetic substrate (N-tert-butoxycarbonyl-Leu-Gly-Arg-p-nitroanilide),followed by freeze-drying. 0.1 ml of 0.2M Tris-HCl buffer (pH 8.0) and0.1 ml of a sample (an aqueous solution of endotoxin or (1→3)-β-D-glucanin various concentrations used in Example 1) were added to the resultingfreeze-dried product and the mixture was incubated at 37° C. for 30minutes. The reaction was terminated by adding 0.4 ml of 0.6M aceticacid and an absorbance at 405 nm was measured. The results are shown inFIG. 4 as well as the results obtained using untreated LAL in the samemanner. In FIG. 4, the symbol ◯ stands for an absorbance obtained in thecase of using untreated LAL and the symbol Δ stands for an absorbanceobtained in the case of using LAL treated with laminaran-immobilizedCellulofine. As is clear from the Figure, LAL treated withlaminaran-immobilized Cellulofine did not react with (1→3)-β-D-glucanbut reacted with endotoxin. Thus, endotoxin can be specificallydetermined using the LAL treated with laminaran-immobilized Cellulofine.

EXAMPLE 3 Preparation of LAL using laminaribiose-immobilized Toyopearl

60 ml (wet volume) of laminaribiose-immobilized Toyopearl) obtained inPreparation Example 16 was successively washed with 100 ml of 0.1M NaOHand 1500 ml of distilled water on a glass filter. A 25-fold dilution of2 ml of untreated LAL with distilled water obtained in the same manneras in Example 2 was added thereto, followed by agitation at 4° C. for 30minutes. The mixture was subjected to a centrifugation (3000 rpm, 10minutes). The supernatant thus obtained was freeze-dried, to whichdistilled water was added to make the total volume 2 ml. To a 0.1 mlportion of the same was added 0.1 ml of various concentrations of anaqueous solution of endotoxin or(1→3)-β-D-glucan and the mixture wasallowed to react at 37° C. for 60 minutes. As a result, no gelation wasobserved when (1→3)-β-D-glucan (0.1 to 10,000 ng/ml) was added, whilegelation was observed when endotoxin (100 pg/ml or more) was added.

What is claimed is:
 1. A method of preparing limulus amoebocyte lysatesubstantially free from factor G which comprises bringing limulusamoebocyte lysate into contact with an insoluble carrier on which a(1→3)-β-D-glucoside structural portion represented by the followingformula (I) produced by depolymerizing and/or fractionating acarbohydrate chain is immobilized by a method which comprisesepoxy-activating an insoluble carrier with epichlorohydrin orbisoxiranes, reacting the resulting epoxy-activated insoluble carrierwith hydrazine hydrate or a dihydrazide compound to obtain a hydrazinederivative or a dihydrazide derivative and bringing the(1→3)-β-D-glucoside into contact with the hydrazine or dihydrazidederivative: ##STR10## wherein n represents an integer of 2 to
 370. 2. Amethod as claimed in claim 1, wherein the (1→3)-β-D-glucoside structuralportion additionally contains at least one structural unit selected fromthe group consisting of structural units represented by the followingformulae (II), (III), (IV), (V) and (VI): ##STR11## wherein at least oneof R₁, R₂ and R₃ represents a group selected from the group consistingof a methyl group, a hydroxyalkyl group, a carboxyalkyl group, an acetylgroup, a sulfate group, a phosphate group, an allyl group, a metal saltof any of said groups, an ammonium salt of any of said groups, and anorganic amine salt of any of said groups, and the remaining groupsrepresent a hydrogen atom.
 3. A method as claimed in claim 1 or 2,wherein the (1→3)-β-D-glucoside structural portion is derived fromnatural sources selected from the group consisting of (1→3)-β-D-glucanderived from bacteria of the genus Alcaligenes, paramylon derived fromEuglena, β-glucan or callose derived from higher plants, laminatansderived from brown algae, chrysolaminarans derived from diatoms,pachyman derived from Poria, glucan derived from the cell wall ofPhytophthora, sclerotan derived from Sclerotinia, schizophyllan derivedfrom Schizophyllum, grifolan LE derived from Grifola frondosa,scleroglucans derived from Sclerotium, Corticium or Stromatinia,lentinan derived from Lentinus, β-glucan derived from the cell wall ofSaccharomyces (bakers' yeast), lichenans derived from Cetraria, Usnea orEvernia, and β-glucan contained in barley endosperm.
 4. A method asclaimed in claim 3, wherein the (1→3)-β-D-glucoside structural portionis produced by depolymerizing a carbohydrate chain by a partialdecomposition method selected from the group consisting of a hydrolysisusing an acid, an alkali or β-glucanase, an acetolysis and a sonication,and/or fractionating a carbohydrate chain by a fractional precipitationmethod using an organic solvent or salts, or a fractionation methodusing a molecular sieve agent or a molecular sieve membrane.
 5. A methodas claimed in claim 1, wherein the insoluble carrier is selected fromthe group consisting of cellulose, agarose, crosslinked dextran,polyacrylamide, porous glass, and a hydrazine or hydrazide derivative ofa synthetic hydrophilic polyvinyl polymer.